The mucosal mast cell line RBL-2H3 secrete histamine, serotonin (5-hydroxytryptamine, 5-HT) and other inflammatory mediators in response to applied allergen. Previously it has been shown that 5-HT distributions can be imaged in 5-HT loaded RBL-2H3 cells using three photon excitation (3PE) of 5-HT autofluorescence (Maiti et al., 1997, Science 275:530). In this application of multiphoton microscopy, an infrared (740 nm), ultrafast (pulsed) laser was used to excite the < 300 nm 5-HT electronic transition, alleviating chromatic aberrations and the severe photodamage associated with deep UV wavelengths. We are making 3PE imaging measurements of RBL-2H3 cell secretion. During imaging the cells are observed to characteristically ruffle and spread as granular 5-HT autofluorescence disappears with no detectable granule movement. Individual cells exhibit highly heterogeneous release kinetics, often with quasi periodic bursts. Neighboring granule disappearances are generally correlated, indic ative of either spatially localized signaling or granule-granule interactions. After individual granule release events, weak residual fluorescence patterns retaining the initial granule shapes are often visible, suggesting leftover 5-HT still bound to the granule matrix. In two-color experiments, we are using a LysoSensor dye to simultaneously record the pH history of granules as they secrete. Using this dye pH jumps are observed and we are presently examining their temporal relation to calcium oscillations that also occur upon cell stimulation.